Fluorescence detection

ABSTRACT

The present invention provides methods of detecting and/or characterizing the viral vector particle content of a medium. A medium is provided and contacted with an excitation energy such that, if a viral vector particle is in the medium, an electron associated with the intrinsically fluorogenic portion of the viral vector particle will be raised to an excited energy state. The excited electron is permitted to emit radiation having an emission wavelength which is detected. The viral vector particle content of the medium then can be evaluated by comparing the detected emission wavelength with a standard signal. For example, the number of viral vector particles in a medium can be quantified by comparing the detected wavelength and its corresponding intensity to a standard signal. Similar methods for evaluating the adenoviral vector particle content of a medium and the intrinsically fluorogenic adenoviral structural protein content of a medium are provided.

This is a divisional of application Ser. No. 09/679,439, filled on Oct.4, 2000, which is incorporate by reference.

TECHNICAL FIELD OF THE INVENTION

This invention pertains to the detection and characterization of viralvector particles, particularly through the use of fluorescence.

BACKGROUND OF THE INVENTION

Viral vectors are of significant importance in several aspects ofmolecular biology and medicine. Numerous types of viral vectors havebeen developed for use as gene delivery vehicles. Examples of such viralvectors include vectors based on adenovirus (Ad), adeno-associated virus(AAV), baculovirus, herpes simplex virus (HSV), and murine leukemiavirus (MLV). Compared to other methods of delivering genetic information(e.g., lipo some-associated delivery techniques or naked DNA vectors),viral vectors offer several advantages, including higher rates ofdelivery and better targeting of specific tissues and/or cells. With theincreased use of viral vectors for therapeutic, as well as diagnostic,applications there is an increasing need for better methods forquantification and characterization of viral vector particles.

Several techniques are known for the characterization and/orquantification of viral vector particles, including chromatographicmethods and mass spectrometry (see, e.g., International PatentApplication WO 99/54441, International Patent Application WO 00/40702,and U.S. Pat. No. 5,965,358). Presently, the quantification of viralvector particles is most commonly carried out by the use of ultraviolet(UV) radiation. For example, U.S. Pat. No. 5,837,520 disclosesmonitoring the absorbance of a chromatographic eluant of viral particlesat a selected UV wavelength and comparing the absorbance value to astandard curve which relates absorbance to the number of viral vectorparticles. Ultraviolet absorbance is limited in its sensitivity andrequires a large number of viral particles (typically about 5×10⁹particles/ml) for accurate detection (where the standard deviation inmeasurement is about 10% or less). Due to the large number of viralparticles required for accurate quantification, ultraviolet absorbanceis not useful in applications requiring small populations of viralparticles, such as viral vector-based gene therapies where highquantities of viral vector particles can be undesirable.

Fluorescence-based detection and quantification of viral particlesassociated with fluorogenic dyes such as fluorescein isothiocynate(FITC) is known in the art. For example, Hara et al., Applied andEnvironmental Microbiology, 57(9), 2731-34 (1991), describes the use ofepifluorescent microscopy on DAPI(4′,6′-diamidino-2-phenylindole)-treated water samples to determinenumbers of bacteria, viruses, and DNA-associated particles. Morerecently, Hennes and Suttle, Limnol. Oceanogr., 10(6), 1050-55 (1995),describes similar research using the cyanine-based dye, Yo-Pro-1.

Immunofluorescence, which combines antibody-antigen binding andfluorophore-associated fluorescence detection (see, e.g., Tanaka et al.,J. Hepatology, 23, 742-45 (1995)), also has been used to detect and/orcharacterize viruses. For example, D'alessio et al., AppliedMicrobiology, 20(2), 233-39 (1970), discloses the use offluorescein-based immunofluorescence techniques to detect influenzaviruses, herpes simplex virus, and adenoviruses. More recently, Orito etal., Gut, 39, 876-880 (1996), described the use of a fluorescent enzymeimmunoassay (FEIA) to quantify hepatitis C virus core protein levels inpatients, and Wood et al., J. Medical Virol., 51, 198-201 (1997),describes the use of FITC-based immunofluorescence to identify and typeadenovirus isolates. Enzymatic techniques associated with fluorogenicdyes also are capable of detecting nucleic acids (see, e.g., U.S. Pat.No. 5,830,666).

The Green Fluorescent Protein (GFP), obtained from the jellyfishAequorea victoria (see, e.g., Prasher et al., Gene, 111, 229-33 (1992)),which is intrinsically fluorogenic, has been used to characterizeviruses by causing viruses to express GFP. For example, InternationalPatent Application WO 00/08182 describes preparations of herpes virusexpressing GFP fusion proteins to detect the progress of cell infectionby the virus and to screen for neutralizing antibodies or inhibitors ofinfection. International Patent Application WO 99/54348 discloses theuse of vectors transfected with short-lived GFP variants to assayactivation or deactivation of promoters. International PatentApplication WO 99/43843 teaches transfection with adenovirus vectorsencoding GFP and tracking viral production by GFP-associatedfluorescence.

Techniques for detecting or characterizing viral vector particles basedon direct fluorescent dye-association with the viral particles,immunofluorescence, and GFP-associated viral fluorescence are limited inrequiring either a fluorogenic dye or GFP to be associated with theviral vector particles. Because the use of fluorogenic dyes can beexpensive, less sensitive than other techniques, and damaging tosamples, direct dye-association techniques are often unsuitable.Immunofluorescence, while more sensitive than direct dye-basedtechniques, requires specific epitopes and antibodies. GFP-basedtechniques require either chemical or genetic modification to associatethe viral vector particles with GFP.

Few fluorogenic methods have been used to study biological materialswithout the use of dyes or strong fluorogenic proteins such as GFP. U.S.Pat. No. 5,623,932 discloses the use of direct fluorogenic methods todifferentiate between normal and abnormal cervical tissues. The '932patent discloses using laser-induced fluorescence (LIF) to identifyfluorogenic spectra associated with healthy tissue, relying onoxy-hemoglobin and NADH in the tissue as fluorophores, and further usingsuch spectra to identify “abnormal” tissues by comparing spectra. The'932 patent suggests that such abnormal tissue could be inflamed orinfected with human papilloma virus (HPV). However, the '932 patentfails to identify, characterize, or quantify HPV particles in suchtissues.

Accordingly, there remains a need for techniques which allow forimproved detection and characterization of viral vector particles. Thepresent invention provides methods for such detection andcharacterization through fluorescence detection of viral vectorparticles and viral vector proteins. These and other advantages of thepresent invention, as well as additional inventive features, will beapparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method of quantifying the number ofviral vector particles in a medium. A medium containing a viral vectorparticle having an intrinsically fluorogenic portion is provided. Themedium is contacted with an excitation energy, such that an electronassociated with the intrinsically fluorogenic portion of the viralvector particle is raised to an excited energy state. The excitedelectron is permitted to emit radiation having one or more emissionwavelengths and corresponding emission intensities. An emissionwavelength, and the intensity of the emitted radiation at the emissionwavelength, are detected. The number of viral vector particles in themedium is quantified by evaluating the detected wavelength and intensityand comparing them to a provided standard signal.

The invention also provides a method of evaluating the viral vectorparticle content of a medium. A medium is provided and contacted with anexcitation radiation having an excitation wavelength such that if aviral vector particle is in the medium an intrinsically fluorogenicportion of the viral vector particle will emit radiation having anemission wavelength at about 560-590 nm (e.g., about 575 nm). The viralvector particle content of the medium is evaluated by determiningwhether the medium emits radiation at about 560-590 nm.

The invention further provides a method of evaluating the adenoviralvector particle content of a medium. A medium is provided and contactedwith an excitation radiation having one or more excitation wavelengthssuitable for exciting an electron associated with the intrinsicallyfluorogenic portion of an adenoviral vector (typically at about 235 nm,about 284 nm, or both). If an adenoviral vector particle is in themedium, an intrinsically fluorogenic portion of the adenoviral vectorparticle will emit radiation having an emission wavelengthcharacteristic of a naturally-occurring (i.e., wild-type) adenoviralvector (typically at about 330 nm, about 574 nm, or both). Theadenoviral vector particle content of the medium is evaluated bydetermining whether the medium emits radiation having such an emissionwavelength (or wavelengths).

The invention also provides a method of evaluating the intrinsicallyfluorogenic adenoviral structural protein content of a medium. Similarto the other aspects of the invention, a medium is provided andcontacted with an excitation radiation having an excitation wavelength,such that if an intrinsically fluorogenic adenoviral structural proteinis in the medium it will emit radiation having an emission wavelengthcharacteristic of an intrinsically fluorogenic wild-type adenoviralstructural protein or a substantial homolog thereof. The intrinsicallyfluorogenic adenoviral structural protein content of the medium isevaluated by determining whether radiation having an emission wavelengthcharacteristic of an intrinsically fluorogenic wild-type adenoviralstructural protein or substantially homologous protein is emitted fromthe medium.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of detecting and/orcharacterizing (e.g., quantifying) the viral vector particle content ofa medium. A medium in the context of the present invention is any mediumwhich is suitable for detection of radiation emitted from a wild-typeintrinsically fluorogenic portion (or portions) of a viral vector, orsubstantial homolog thereof, using the disclosed inventive methods. Themedium can include different types of intrinsically fluorogenic viralvector particles, other fluorogenic molecules and non-fluorogenicmolecules. The medium can take any suitable form. Typically, the mediumwill include or be in the form of a liquid, such as an aqueous solution.Such solutions can consist of numerous additional components, such asbuffers, stabilizers, preservatives, excipients, carriers, diluents, orother additives. The medium can comprise a pharmaceutically acceptable(e.g., a physiologically acceptable) carrier and can take the form of apharmaceutical composition. The medium can include one or more cells.For example, the medium can be a culture of cells, or a tissue, which iseither in a tissue culture or in an animal (e.g., an organ in a human).The medium can consist of a sample of a larger composition, such as apool or stock of viral vector particles (e.g., a library of viral genetransfer vector particles in a stock).

The medium can, and typically will, contain a viral vector particlehaving an intrinsically fluorogenic portion. The invention can bepracticed with any suitable type of viral vector particle. A viralvector particle is any molecule which is based upon, derived from, ororiginates from a virus, and which includes more than one type of viralmolecule (e.g., more than one type of viral protein or a viral proteinand a viral nucleic acid) or a substantial homolog thereof (as definedfurther herein). A viral molecule is any molecule which makes up aportion of a wild-type virus or a substantial homolog thereof.

The viral vector particle can be an unmodified naturally occurring(i.e., “wild-type”) virus particle, or modified viral vector particle,such as a viral gene transfer vector and/or a synthetic viral vectorparticle. Desirably, the viral vector particle contains, or isassociated with, a nucleotide genome. Preferably, though notnecessarily, the viral vector particle is derived from, is based on,comprises, or consists of, a virus which normally infects animals, suchas mammals and, especially, humans. Preferred types of viral vectorparticles include baculovirus vectors, herpes vectors, retroviralvectors, adeno-associated viral vectors, and adenoviral vectors.Adenoviral vector particles are particularly preferred.

The inventive method can be practiced with a medium containing anysuitable number of viral vector particles. A suitable number of viralvector particles is any number which can be detected and/orcharacterized (e.g., quantified) by the methods of the presentinvention. The inventive methods can be practiced with a homogenous orheterogeneous (i.e., mixed) population of viral vector particles (e.g.,wild-type herpes virus and adenovirus particles, or different modifiedparticles such as replication defective adenoviral vector particles andcomplementing (i.e., helper) adenovirus particles). When a number ofidentical or similar viral vector particles are present in a suitablemedium, the particle-containing medium can be referred to as a stock ofthe viral vector.

The viral vector particle can have any suitable size and weight. Incontrast to UV spectrophotometry-based techniques, viral vectorparticles with relatively larger molecular weights and sizes can bedetected, characterized, and/or quantified directly using the methodsdescribed herein, without performing calculations or taking additionalsteps to account for scattered light problems associated with UV-baseddetection which may result in erroneous detection readings. For example,viral vector particles having molecular weights of about 1×10⁸ Daltonsor more, about 1.5×10⁸ Daltons or more, and even about 1.7×10⁸ Daltonsor more (e.g., about 2×10⁸ Daltons or more) can be detected and/orcharacterized (e.g., quantified) directly (i.e., without takingadditional steps or performing calculations to account for lightscattering). Further in contrast to UV spectrophotometry-basedtechniques, viral vector particles with large particle sizes can bedirectly detected and/or characterized (e.g., quantified). For example,viral vector particles of at least about 40 nm in diameter, at leastabout 80 nm in diameter, at least about 120 nm in diameter, or larger,can be directly detected and/or characterized.

The viral vector particle includes an intrinsically fluorogenic portion.The intrinsically fluorogenic portion in the context of the presentinvention is any portion of a viral vector particle which includes, orconsists of, a naturally-occurring (wild-type) viral molecule (e.g., anintrinsically fluorogenic viral protein), or a substantially homologous(preferably substantially identical) molecule, which is intrinsicallyfluorogenic. A molecule is “intrinsically fluorogenic” if it emits oneor more emission wavelengths when contacted with a suitable excitationenergy in the absence of fluorescent dyes and/or conjugatedfluorophores.

Typically, and preferably, the intrinsically fluorogenic portionincludes, or consists of, a wild-type viral molecule. In such aspects,the molecule can be any suitable type of molecule. Examples of suitablemolecules include viral proteins, including post-translationallymodified viral proteins (e.g., viral glycoproteins).

The intrinsically fluorogenic portion can include, or consist of, anintrinsically fluorogenic molecule that is at least substantiallyhomologous, preferably substantially identical, to an intrinsicallyfluorogenic wild-type viral molecule (e.g., a non-wild-type homolog of awild-type viral fluorogenic protein). As used herein, a substantiallyhomologous molecule is any molecule having at least about 70% amino acidsequence homology to another molecule (e.g., a wild-type viral protein),at least about 70% structural similarity to another molecule (e.g., awild-type viral protein), or both.

The intrinsically fluorogenic portion has at least about 70% (e.g., atleast about 80%, at least about 85%, or at least about 90%) amino acidsequence homology to an intrinsically fluorogenic wild-type viralmolecule if at least about 70% of the amino acid residues in thesubstantially homologous molecule's amino acid sequence are identicalto, or differ by only conservative amino acid residue substitutionsfrom, the amino acid residues in the amino acid sequence of itswild-type counterpart when the sequences are compared in a manner whichmaximizes homology and/or identity. Conservative amino acid residuesubstitutions involve exchanging a member within one class of amino acidresidues for a residue that belongs to the same class. Homologousproteins obtained by conservative substitutions are expected tosubstantially retain the biological properties and function of thewild-type protein. The classes of amino acids and the members of thoseclasses are presented in Table 1.

TABLE 1 Amino Acid Residue Classes Amino Acid Class Amino Acid ResiduesAcidic Residues ASP and GLU Basic Residues LYS, ARG, and HIS HydrophilicUncharged Residues SER, THR, ASN, and GLN Aliphatic Uncharged ResiduesGLY, ALA, VAL, LEU, and ILE Non-polar Uncharged Residues CYS, MET, andPRO Aromatic Residues PHE, TYR, and TRP

A substantially homologous molecule can include any suitable number ofnon-conservative amino acid residue substitutions. Preferably, aromaticresidues (which are fluorogenic) remain conserved with respect to, andmore preferably remain identical to, the aromatic residues occurring inthe corresponding wild-type viral molecule.

One of ordinary skill will recognize that residue position in eithersubstantially homologous or substantially identical molecules may varyfrom their wild-type counterpart molecule due to deletions or additionsof residues. Homology and/or identity in view of such substitutions anddeletions can be determined using commercially available sequenceanalysis/alignment software and/or other known techniques. Protean, soldby DNASTAR (Madison, Wis.), is an example of suitablecommercially-available sequence analysis software.

Alternatively or additionally, the intrinsically fluorogenic portion hasat least about 70% (e.g., at least about 80%, at least about 85%, or atleast about 90%) structural similarity to an intrinsically fluorogenicwild-type viral molecule. Thus, the intrinsically fluorogenic portioncan have a significantly different amino acid sequence from wild-typeviral proteins if there exists such structural similarity. For example,the invention can be practiced with synthetic peptides and/orrecombinantly produced peptides that have a structure that issubstantially similar to the structure of wild-type adenovirus fiberprotein. Examples of such proteins include modified fiber proteins thatcontain knob (or “head”) region or domain modifications as described in,e.g., U.S. Pat. No. 5,846,782, and double-ablated adenoviruses thatcontain modified penton proteins.

The percentage of structural similarity can be based on complete overlapbetween the molecules, on a domain-by-domain basis, or, preferably, byboth methods. Structural similarity between the molecules can bedetermined by any suitable method. For example, the secondary structureof two proteins can be determined and compared, e.g., by means ofperforming surface probability comparisons using the amino acidsequences of both molecules. Alternatively, and preferably, the threedimensional structures for the two proteins are determined and compared(e.g., by overlapping the three dimensional structures of the proteinsusing three dimensional imaging software).

The intrinsically fluorogenic portion can be substantially identical toan intrinsically fluorogenic wild-type viral molecule such that it hasat least about 70% (preferably at least about 80%, and more preferablyat least about 90%) amino acid sequence identity with a wild-type viralprotein. Preferably, although not necessarily, the intrinsicallyfluorogenic portion also will have at least 70% structural similarity toits wild-type viral counterpart.

The intrinsically fluorogenic portion desirably has at least similarfluorogenic properties to its wild-type viral counterpart. In otherwords, the intrinsically fluorogenic portion preferably emits radiationhaving at least one emission wavelength in common with its wild-typeviral counterpart.

The viral vector particle can include, and preferably does include, morethan one intrinsically fluorogenic portion, and each intrinsicallyfluorogenic portion preferably includes more than one intrinsicallyfluorogenic molecule (e.g., 3, 5, 10, 15, or more intrinsicallyfluorogenic wild-type viral proteins). Additionally or alternatively,the viral vector particle can include non-natural and/or non-viralfluorogenic portions in addition to the intrinsically fluorogenicportion (e.g., the viral vector particle can include GFP).

The intrinsically fluorogenic portion can make up any suitable portionof the viral vector particle, including the entire particle. Desirably,the molecules which make up the intrinsically fluorogenic portion (orportions) make up at least about 10%, preferably at least about 20%,more preferably at least about 50%, and even more preferably at leastabout 70% of the molecules which form the viral vector (either byweight, molecule type, or both).

The viral vector particle-containing medium is contacted with anexcitation energy. The excitation energy can be any form of energy whichis capable of exciting an electron associated with an intrinsicallyfluorogenic portion of the viral vector particles to an excited energystate. While numerous forms of energy are suitable, the excitationenergy preferably is in the form of an excitation radiation.

Excitation radiation can be any suitable type of radiation. Typically,the excitation radiation will be in the form of electromagneticradiation having one or more discrete wavelengths. For example, theexcitation radiation can be in the form of visible light radiation, suchas is emitted from a suitable lamp. Alternatively, the excitationradiation can be in the form of ultraviolet radiation (UV) or infrared(IR) radiation. The excitation radiation can be generated by anysuitable technique or device. Most often, the excitation radiation is inthe form of one or more photons of energy supplied by a suitableradiation source, such as an incandescent lamp, argon/mercury lamp,xenon lamp, halogen lamp, or a laser (e.g., through a laser-inducedflash).

Due to the speed with which excitation occurs using a xenon lamp or alaser, the excitation energy is preferably provided by one of these twosources. Because of its high sensitivity, laser-induced fluorescence(LIF) is particularly preferred. In LIF, the medium typically isirradiated at one wavelength, usually in the UV spectral region, and theemission (fluorescent signal) is measured at a longer wavelength,usually at a higher UV wavelength or the violet-yellow/green region ofthe visible spectrum. The excitation source for molecular LIF typicallyis a tunable dye laser in the UV spectral region. In addition to UVradiation, LIF can utilize visible and/or near-IR excitation radiation,particularly with recently developed frequency doubling methods. Coolingof the medium (and thus the viral vector particle), for example bymolecular beams, free-jet expansions, and cryogenic glass or crystallinematrices, in LIF-based techniques, can remove spectral congestion andreduce the Doppler width of the transitions, thereby allowing forimproved detection. The laser used for LIF can be any suitable laser,including, for example, an argon-ion laser or a helium/neon laser.Preferred laser fluorometers are ZetaLIF fluorometers, available fromPicometrics (Ramonville, Saint Agne, France). Typically, in using LIFtechniques, the medium will be, or comprise, a sample (i.e., a portion)of a larger composition due to the tendency of LIF techniques to damagebiological samples, including viable viral vector particles. Due to thehigher sensitivity associated with LIF-based techniques, such samplescan be relatively small and consist of very few viral vector particles(e.g., samples containing about 1×10⁶ viral vector particles or less aresuitable), and the results of applying the method can be extrapolated toquantify or otherwise characterize or evaluate the viral vectorparticles in a significantly larger composition.

An electron associated with an intrinsically fluorogenic portion of theviral vector particle is raised to an excited energy state by thecontact of the excitation energy with the medium. The process of raisingthe electron to an excited energy state is known as excitation, and theelectron in such a state is referred to as an excited electron.Excitation in the context of the present inventive method can occur inany suitable manner. For example, excitation can occur through directcontact of the viral vector particle with the excitation energy, or,alternatively, through absorption of the excitation energy and transferthereof through the medium to the viral vector particle. Any suitablenumber of electrons can be raised to the excited state by the excitationenergy. Desirably, more than one electron is excited. Thus, each viralvector particle desirably includes an intrinsically fluorogenic portionassociated with more than one excited electron.

The excited energy state can be any suitable energy state which ishigher than the energy state which the electron occupied immediatelyprior to contact with the excitation energy. A suitable energy state isany energy state which causes the excited electron, if permitted, toemit radiation. Thus, the excited electron can be raised (i.e.,“boosted”) to the next highest energy state it can occupy (a firstexcited state (e.g., an S₁ state)), or to a higher energy state whichthe electron can occupy (a second or higher excited state (e.g., an S₂state)). An electron's energy state can be characterized based on thevibrational energy, the rotational energy, or both, associated with theenergy state. Several vibrational and rotational energy levels can existwithin an excited state.

A radiation wavelength associated with exciting an electron is anexcitation wavelength. The excitation wavelength associated with aparticular viral vector particle is dependent upon the fluorogenicproperties of the particular viral vector. The radiation wavelengthassociated with exciting the largest number of viral vector particles isthe optimum excitation wavelength. The optimum excitation wavelength canbe determined by determining the excitation wavelength associated withthe apex of the highest “peak” on a graph of the viral vector particle'sexcitation spectrum (i.e., a two-dimensional plot of either excitationenergies or wavelengths versus the intensity of the resulting emittedradiation).

The viral vector particle can have any suitable number of associatedexcitation wavelengths. Preferably, the viral vector particle has morethan one associated excitation wavelength. In such situations,quantification of the number of viral vector particles typically ispracticed using the optimum excitation wavelength, which provides thegreatest sensitivity and most accurate detection of viral vectorparticles. In some situations, however, using other excitationwavelengths is preferred. For example, an excitation energy associatedwith the viral vector particles, which does not excite other (i.e.,non-viral vector) fluorogenic components of the medium, can be used toprovide greater selectivity for the viral vector particles, even if theexcitation radiation is not the optimum excitation wavelength.

Excitation of other fluorogenic components of the medium also can beavoided by the use of wavelength selectors, which are known in the art.A wavelength selector screens radiation, thereby permitting only certainwavelengths, or bands (i.e., ranges) of wavelengths, to contact themedium. Any suitable type of wavelength selector can be used. Typicalwavelength selectors include monochromators, bandpass filters (such aslong pass and short pass filters), and cutoff filters. A monochromatoror a bandpass filter permits a range of wavelengths of an excitationradiation to pass through and contact the medium while blockingradiation at the other excitation wavelengths. A monochromator increasesthe intensity of the resulting fluorescent emissions by selecting for arange of excitation wavelengths. A cutoff filter blocks stray excitationradiation below a predefined cutoff point. Monochromators, bandpassfilters, and other components can be included within a fluorescencedetection system. For example, the system can include one or moregratings which are designed to optimize excitation and/or emissionwavelengths, alone or in combination with one or more mirrors fordirecting excitation radiation to the medium or a portion thereof.

After excitation, the excited electron, if permitted to, will emitradiation having one or more emission wavelengths. This phenomenon ofemitting radiation by an excited electron concomitant with the excitedelectron's return to a ground or relaxed state is known as fluorescence.The excited electron's emission of radiation (also known as afluorescent emission) permits the excited electron to enter an energystate lower than the excited energy state (sometimes referred to as theground or relaxed state), which typically is substantially equal to theenergy state the electron was in prior to contact with the excitationenergy.

Fluorescent emissions are marked by brief excitation emission periods.Fluorescent emissions usually begin almost instantaneously uponabsorption of radiation at a suitable excitation wavelength. Afterexcitation, fluorescent emissions can occur for any suitable period.Preferably, fluorescent emissions occur for about 5×10⁻³ seconds orless. Typically, fluorescent emissions will occur within a period ofabout 1×10⁻⁵−1×10⁻⁹ seconds.

The emitted radiation can have any suitable number of emissionwavelengths. The magnitude of the emission wavelengths is dependent uponthe excitation wavelength and fluorogenic characteristics of the viralvector particle, particularly the energy levels available to the viralvector particle-associated excited electrons. Similar to excitationwavelengths, emission wavelengths can form an emission spectrum, whichcan be graphically represented as a plot of emission wavelengths versusfluorescence intensity.

Because of energy dissipation during absorption, the emission wavelengthor wavelengths typically are longer than the excitation wavelength orwavelengths. The difference in energy or wavelength represented by thedifference between the excitation wavelength and the emission wavelength(hν_(EX)-hν_(EM)) is known as the Stokes shift. This difference inlength of the excitation and emission wavelengths, represented by theStokes shift, permits isolation of either excitation or emissionradiation. Accordingly, viral vector particles associated with largeStokes shifts are preferred.

Fluorescent emissions, in the context of the present invention, can haveany suitable characteristics. Preferably, the fluorescent emissions aredistinguishable from phosphorescent emissions or luminescent emissions.Thus, for example, the production of fluorescent emissions compared tothe use of UV spectrophotometry is relatively temperature independent,except with regard to an increased probability of quenching associatedwith higher temperatures in some mediums.

The fluorescence process usually is cyclical. Thus, unless the intrinsicfluorogenic capacity of a viral vector particle is irreversiblydestroyed in the initial excited state (for example by the phenomenon ofphotobleaching), a viral vector particle can be repeatedly excited anddetected. Thus, the excitation of an electron associated with a viralvector particle can be repeated as desired depending upon the ability ofthe viral vector particle to undergo repeated excitation/emissioncycles.

Often, not all of the electrons initially excited return to the lowerenergy state by fluorescence. Other processes such as collisionalquenching, fluorescence energy transfer, and intersystem crossing alsocan depopulate the population of excited electrons. The ratio of thenumber of fluorescence emissions to the number of photons absorbed by aviral vector particle is the fluorescence quantum yield. Thus, thequantum yield measures the relative extent to which processes whichdepopulate the population of excited electrons occur.

In order to maximize the quantum yield, the methods of the presentinvention preferably are practiced while avoiding photobleaching (i.e.,photodestruction) and quenching of the fluorogenic properties of theviral vector particle. Any suitable technique for avoidingphotobleaching and quenching can be utilized. Examples of suitabletechniques include avoiding high intensity excitation radiation,maximizing detection sensitivity (e.g., by using low-light detectiondevices such as CCD cameras, as well as high-numerical apertureobjectives, and the widest emission bandpass filters compatible withsatisfactory signal isolation), using antifade agents, and avoidingagents associated with collisional quenching, such as O₂ and heavy atomssuch as iodide. When the medium is in solution (e.g., a portion of acomposition subjected to a chromatography resin), degassing thecomposition to remove such agents, particularly oxygen, and therebyavoid collisional quenching, is particularly preferred. In livecell-containing mediums, vitamin C (ascorbic acid) often can be used toreduce photobleaching. Collisional and self-quenching also can often bereduced, if necessary, by reducing the concentration of the viral vectorparticles and/or other components in the medium. Quenching isconformation dependent. Thus, modifying the conformation of the viralvector particles also can affect the probability of quenching.Environmental factors, such as medium polarity, proximity andconcentrations of quenching species, and pH, also should be monitoredfor photobleaching effects.

The emitted radiation can be characterized on the basis of its intensity(also sometimes referred to in the art as brightness). The totalintensity of viral vector particle emitted radiation in a particularmedium is a function of the intensity and wavelength of the excitationradiation, the amount of viral vector particles present in the medium,and the fluorogenic properties of the viral vector particles.

Two fluorogenic properties of the viral vector particles which affectintensity are the extinction coefficient and quantum efficiency. Theextinction coefficient is the amount of radiation of a given wavelengththat is absorbed by the viral vector particle upon contact with theexcitation radiation. The quantum efficiency of the viral vectorparticle is its capacity to convert such absorbed radiation to emittedfluorescent radiation. The molar extinction coefficient of a viralvector particle is defined as the optical density of a one-molarsolution of viral vector particles through a one-cm radiation path.Emission intensity is proportional to both the quantum efficiency andextinction coefficient.

The emitted radiation, particularly the wavelength and intensity of theemitted radiation corresponding to at least one emission wavelength, isdetected. If the viral vector particle emits radiation at multipleemission wavelengths, it is typically preferred that the method includesdetermining the intensity of emitted radiation at those wavelengths aswell. Any suitable number of emission wavelengths, and intensitiescorresponding to any number of emission wavelengths, can be detected.

The emitted radiation can be detected using any suitable technique.Preferably, a fluorescence detector is used to detect the emittedradiation. Any suitable fluorescence detector can be used, and numeroustypes are known and commercially available. Generally, a fluorescencedetector registers emission radiation, including emission wavelengths,intensities, or both, and produces a recordable output, usually as anelectrical signal or a photographic image. To aid detection, thefluorescence typically interacts with an emission wavelength selector(such as a monochromator or an interference filter) and then is detectedby a radiation detector, such as a photodiode or a photomultiplier tube(PMT). Other radiation detectors, such as a CCD camera, also can beused.

Suitable fluorescence detectors include fluorometers (sometimes referredto in the art as fluorimeters), spectrofluorometers, and microplatereaders, which measure the average fluorescent properties of the medium;fluorescence microscopes, which resolve fluorescence as a function ofspatial coordinates in two or three dimensions; fluorescence scanners,which resolve fluorescence as a function of spatial coordinates in twodimensions for macroscopic objects such as electrophoresis gels, blots,and chromatograms; and flow cytometers, which measure fluorescence perparticle in a flowing stream, allowing subpopulations of viral vectorparticles in the medium to be identified, evaluated, and quantified.Each type of instrument produces different measurement artifacts andmakes different demands on the viral vector particles. For example,although photobleaching is often a significant problem in fluorescencemicroscopy, it is not a major impediment in flow cytometry because thedwell time (how long the excitation beam continues to illuminate themedium) of the individual viral vector particles cells in the excitationbeam in flow cytometry is short. PMTs can be useful in low intensityapplications such as fluorescence spectroscopy and are often integratedinto such devices; however, other radiation detectors also are suitable.

Preferably, the fluorescence detector provides continuous ranges ofexcitation and emission wavelengths, in contrast to laser scanningmicroscopes and flow cytometers, which presently typically requireexcitation at a single fixed wavelength. Fluorometers and/orspectrofluorometers, which provide such qualities, are preferredfluorescence detectors. Generally, a fluorometer is a fluorescencedetector which includes an excitation source, a sample cell for testingthe medium (which typically is a portion of a larger composition), and aradiation detector, such as a PMT.

As indicated above, scanning fluorescence techniques are useful in manyaspects of the invention. Examples of such techniques include moving alaser over the medium or, alternatively, using a CCD camera to collectthe entire image at once. CCD camera techniques are faster andpotentially more sensitive than scanning, but often provide lowerresolution than PMT-based scanning techniques. Other detectionconfigurations have been developed using multiple lasers, rotatingmirrors, and mounts that fix the laser and detectors in a constantposition, each of which provides different and particular advantages.For example, certain configurations can permit the determination of theshape and/or weight of the viral vector particle (e.g., systems whichuse a fluorometer similarly to a light scatter detector).

Detecting fluorescent emissions sometimes can be compromised bybackground signals, which may originate from other medium constituents(sometimes referred to in the art as autofluorescence). Autofluorescencedesirably is minimized. Autofluorescence can be minimized by using asuitable wavelength selector, such as a filter that reduces thetransmission of background signals (e.g., a bandpass filter).Alternatively, in three-dimensional imaging systems, confocal opticsimprove resolution in the third dimension. Such systems irradiatesequentially each point in three-dimensional space. Collection opticscollect the signal from the irradiated point and reject any informationthat is out of focus. If the viral vector particles are associated withmultiple excitation wavelengths, the use of longer wavelengths also canassist in avoiding background fluorescence. Another way to improve thesignal is to increase the viral vector particle concentration; however,in most instances care should be taken to avoid quenching caused at veryhigh viral vector particle density.

There are numerous other ways to improve signal detection andevaluation. For example, the excitation radiation can be eliminated fromthe collection pathway by several methods, including orienting the pathof the excitation radiation so that the excitation radiation avoidscontacting the detection pathway and inserting bandpass filters into thedetection pathway to reject the excitation wavelength. Fluorescentsignal strength also can be improved by increasing the dwell time orrepetitively scanning the sample and mathematically processing thesignals to reduce random noise.

A standard signal can be provided, and the number of viral vectorparticles in the medium can be quantified, by comparing the intensity ofthe detected fluorescent emissions emanating from the viral vectorparticle or particles with the standard signal. The intensity of theradiation emanating from the viral vector particle or particles istypically proportional to the number of viral vector particles emittingradiation, thereby permitting relative quantification of the viralvector particles by comparison to the standard signal. In practice, aradiation detector (such as a PMT) which transmits a currentproportional to the intensity of the radiation detected by it can beused to determine the intensity.

Quantification can be performed under any suitable conditions. Typicallyand preferably, wavelength and intensity of the excitation radiation areheld constant (for example, using a controlled laser light source) toensure proportionality between intensity and the number of viral vectorparticles. Dwell time also can affect the intensity of the emittedradiation and also should be kept constant when determining intensityfor quantification purposes.

The standard signal can be any suitable signal which permitsquantification of the number of viral vector particles in the medium.There are numerous techniques available for obtaining a suitablestandard signal. For example, a standard medium having a known viralvector particle content can be used to produce a standard signal (e.g.,a standard emission spectrum), which can be compared to the emissionspectrum of the medium.

The inventive method can quantify any suitable number of viral vectorparticles in any suitable concentration. Desirably, the proportionalityof the number of viral vectors to emitted radiation intensity ismaintained throughout a wide range of viral vector particleconcentrations, though this is sometimes not possible at particularlyhigh concentrations. One skilled in the art can determine the suitablequantifiable range of particle concentrations and particle numbers byroutine experimentation. For example, the minimum number of viral vectorparticles for use can be determined by adding viral vector particles toa medium containing no viral vector particles in a stepwise manner, andtesting for fluorescence detection after each addition. The maximumconcentration and/or particle number can be determined by continuing thesteps of stepwise addition of viral vector particles and fluorescencedetection until the substantially linear relationship between viralvector particle number and emitted radiation intensity is no longerobserved. The fluorescence detector, the type of excitation radiation,and type of viral vector particle, may impact on the range of viralvector particle numbers which can be quantified by the inventive method.

The inventive method can be practiced using mediums containingsignificantly smaller viral vector particle populations compared tothose which can be detected by UV spectrometry. For example, mediumswith viral vector concentrations of about 1×10¹⁰ particles/ml or less,about 1×10⁹ particles/ml or less, about 1×10⁷ particles/ml or less,about 5×10⁶ particles/ml or less, about 1×10⁵ particles/ml or less, oreven lower concentrations, can be quantified using the inventive method.The amount of viral vector particles required for quantification dependsupon the source of the excitation energy. For example, using a xenonlamp to generate the excitation energy permits quantification of about1×10⁷ particles/ml or less (e.g., about 5×10⁶ particles/ml or less),whereas using LIF to generate the excitation energy permitsquantification of about 1×10⁵ particles/ml or less (e.g., about 5×10⁶particles/ml or less).

While quantification can be performed with any suitable level ofaccuracy, the present invention offers methods where quantification atsuch low concentrations is possible with relatively (e.g., compared toUV spectrophotometry) high levels of accuracy. For example, the range oferror (or coefficient of variation) in the detected quantity of viralvector particles using the techniques described herein is typicallyabout 15% or less, preferably about 10% or less, even more preferablyabout 5% or less, and optimally about 3% or less.

In some instances, fluorescence detection by UV spectrometry-basedtechniques can be desirable. For example, when the methods describedherein are used to detect viral vector particles by fluorescencedetection and the medium contains a very large population of viralvector particles (e.g., about 1-2×10¹⁰ particles or more), UVspectrometry-based quantification of viral vector particles may bedesirable.

As indicated above, the inventive method can be practiced using mediumsconsisting of a crude cell lysate of viral vector infected cells or withpurified lysates. Purification can significantly improve the accuracy ofquantification and remove improperly processed (i.e., empty ordefective) or otherwise damaged viral vector particles. Any suitabletechnique for purification can be used. Examples of suitablepurification techniques include chromatographic purification (e.g.,anion exchange chromatography purification), filtration purification(e.g., tangential flow ultrafiltration), and density gradientpurification (e.g., cesium chloride (CsCl) density gradientpurification). Such techniques can be combined or repeated as desired.

Purification by chromatography is preferred. Any suitable type ofchromatographic purification can be used. Preferably, chromatographypurification is performed using the anion exchange chromatographymethods described in International Patent Application WO 99/54441.Desirably, the medium is provided by contacting chromatography resinwith a composition comprising a viral vector particle, and eluting atleast a portion of the composition containing the viral vector particlefrom the chromatography resin, such that the time of elution of theviral vector particle from the chromatography resin is determinable. Thetime of elution of the viral vector particle provides another tool forevaluating the viral vector particle content of the medium.Particularly, by separating a viral vector particle containingcomposition on the basis of elution from a chromatography resin andapplying the inventive method to one or more of the portions of theseparated composition (i.e., treating each portion as a separate mediumfor purposes of the inventive method), one can distinguish between theviral vector particle and other fluorogenic components of the mediumexhibiting similar emission wavelengths on the basis of their respectiveelution times. Moreover, when the expected time of elution of the viralvector particle is known, such techniques provide a way to ensure that adetected fluorescent emission is associated with the viral vectorparticle, by comparing the observed time of elution with a standard(e.g., expected) time of elution. In addition, graphing elution timeagainst fluorescence intensity provides an elution spectrum. Suchelution spectrums can be used for relative quantification purposes.Fluorescence detection can be performed directly on the portion(s) ofthe composition suspected of containing the viral vector particle assuch portion(s) elute from the chromatography resin.

Purified mediums, when used, can be purified to any suitable level.Preferably, a purified medium is at least as pure as a lysate of viralvector infected cells subjected to 1×CsCl density gradient purification.More preferably, the medium is at least as pure as a lysate subjected to2×(i.e., twice repeated) CsCl density gradient purification, and evenmore preferably is substantially as pure as a 3×CsCl density gradientpurified lysate. Examples of techniques for achieving high levels ofpurification are described, for example, in International PatentApplication WO 99/54441.

The present invention also provides a method of quantifying the numberof damaged viral vector particles, such as the number of defective viralvector particles, empty viral vector particles, or both, in the medium,by fluorescence detection. Defective viral vector particles are viralvector particles which are incompletely processed (i.e., contain one ormore incompletely processed components such that they are not as intactas a fully processed viral vector particle). Empty viral vectorparticles are particles which do not contain substantially any (e.g.,about 10% or less, more typically about 5% or less, and even moretypically about 1% or less) of their typical nucleic acid content.

Quantification of defective viral vector particles, empty viral vectorparticles, otherwise damaged particles, or any combination thereof canbe performed with any type of viral vector particle that exhibitsdifferent emission spectrums when such particle is empty, defective, orotherwise damaged as compared to a fully intact viral vector particle,i.e., a fully processed viral vector that is not empty, defective, orotherwise damaged. Typically, such viral vector particles exhibit achange in one or more “damage-sensitive” emission wavelengths, such as awavelength shift and/or an intensity shift in the radiation emitted fromsuch particles when excited. Thus, by detecting the shift in intensityand/or wavelength the number of defective, empty, or otherwise damagedviral vector particles, or any combination thereof, can be quantified bycomparison with a suitable standard signal.

A wavelength shift occurs when a damage-sensitive wavelengthcorresponding to a fully intact (undamaged) viral vector particle isreplaced by a slightly larger or smaller wavelength when the inventivemethod is practiced with a medium containing a number of defective,empty, or otherwise damaged viral vector particles. A wavelength shiftcan include any detectable shift in wavelength. Typically, a wavelengthshift will be about 20 nm or less (e.g., about 10 nm or less) inmagnitude.

The number of defective, empty, or otherwise damaged particles desirablyis determined by an intensity shift. An intensity shift occurs when theemitted radiation at a damage-sensitive wavelength has a detectablyhigher or lower intensity when emitted from defective, empty, orotherwise damaged viral vector particle versus when emitted from a fullyintact viral vector particle of the same type of viral vector. Whether awavelength shift, intensity shift, or both is observed when the mediumcontains damaged viral vector particles depends on the particular typeof viral vector particle.

In quantifying defective viral vector particles, the standard signal canbe any suitable standard signal which enables relative determination ofthe quantity of defective, empty, and/or otherwise damaged particles.For example, the standard signal can correspond to a signal producedfrom a medium having a relatively known amount of defective, empty,otherwise damaged viral vector particles, or any combination thereof. Inthat respect, a crude cell lysate of viral vector particles can beenriched as to the number of defective, empty, or otherwise damagedviral vector particles, and a purified stock of viral vector particleswhich contains relatively few, if any, defective, empty, or otherwisedamaged particles, can be can be used to provide standard signals forcomparing emission spectrums obtained from other mediums (e.g., a crudecell lysate of viral vector particles). A linear regression between thedetected intensities at a damage-sensitive wavelength for the purifiedstock and the damage particle-enriched lysate allows for thequantification of the number of defective, empty, or otherwise damagedviral vector particles in mediums containing more defective, empty, orotherwise damaged viral vector particles than the purified stock, butless than the damage particle-enriched lysate.

Quantification of the number of defective, empty, or otherwise damagedviral vector particles in a medium can be performed under any suitableconditions. Preferably, such techniques are performed in a medium whichis at, and which has been maintained at (e.g., stored at for a period ofat least about 1 hour, at least about 12 hours, at least about 24 hours,at least about 1 week, or longer), a substantially constant medium pH,at a substantially constant medium temperature, and free of particleintegrity-degrading detergents, to avoid undesired integrity changes,conformation changes, quenching, and/or photobleaching. For example, theinventive method can be performed with a medium including or consistingof a pharmaceutical composition, maintained under the aforementionedmedium conditions, which comprises a stock of a viral vector, to assesswhether the pharmaceutical composition is suitable for administration(e.g., by examining particle degradation under such conditions).Although particularly desirable in connection with the quantification ofthe number of defective, empty, or otherwise damaged viral vectorparticles in a medium, these conditions also can be useful in connectionwith other aspects of the inventive method concerning the detectionand/or characterization (e.g., quantification) of viral vector particlesin a medium even in the absence of the quantification of the number ofdefective, empty, or otherwise damaged viral vector particles in amedium.

Preferably, the viral vector particle also is associated with anemission wavelength that is relatively insensitive to the number ofdefective, empty, and/or otherwise damaged viral vector particles in themedium. In other words, such a wavelength and/or intensity remainsrelatively unchanged (e.g., less than about 10%, preferably less thanabout 5%, and more preferably less than about 3% changed) by thepresence of defective, empty, or otherwise damaged viral vectorparticles in the medium. The total number of viral vector particles andthe number of defective, empty, or otherwise damaged viral vectorparticles then can be relatively quantified by evaluating the emissionintensity at the insensitive wavelength and comparing it to a standardsignal, doing the same with regard to the emission intensity at thedamage-sensitive wavelength or wavelengths, and comparing the twoobtained values. The ratio of defective, empty, and/or otherwise damagedviral vector particles to the total number of particles thereby can bedetermined.

The inventive method described herein can be used to evaluate a protocolfor the production of a stock of viral vector particles, such as a stockof a viral gene transfer vector. In such respect, a stock of a viralvector, preferably a viral gene transfer vector, is produced inaccordance with a production protocol. The inventive method then isperformed on a medium containing the stock, or a portion thereof. Theproduction protocol is evaluated by quantifying the number of viralvector particles in the medium, the number of defective, empty, orotherwise damaged viral vector particles in the medium, or anycombination thereof.

Production protocols can be evaluated for any suitable quality and inany suitable manner. For example, different viral vector stockproduction protocols can be compared for the total number of viralvector particles produced and/or the number of defective, empty, orotherwise damaged particles produced. Using such techniques, one candetermine the optimum factors for producing a stock, such as whatharvest time is associated with a desired particle yield of total viralvector particles (based on quantity and/or quality of the viral vectorparticles produced). Another example of a quality which can be evaluatedis the consistency of the production protocol.

The inventive method also can be used for evaluating a pharmaceuticalcomposition including a stock of a viral vector, such as a stock of aviral gene transfer vector. The pharmaceutical composition can be anycomposition containing a stock of a viral vector and a suitablepharmaceutical (e.g., physiological) carrier (such as water, with orwithout other additives (e.g., sugars, salts, and buffers)). The numberof viral vector particles in the pharmaceutical composition can bequantified to determine whether the pharmaceutical composition issuitable for administration. For example, whether the dosage is correctcan be evaluated (e.g., whether a desired dose of viral gene transfervector particles is present). Alternatively or additionally, the numberof intact viral vector particles can be determined to assess whether thenumber and/or percentage of intact viral vector particles in thepharmaceutical composition is acceptable for administration to apatient.

The fluorescence detection methods of the invention can be combined withany number of other fluorescence detection techniques. For example, theviral vector particle can be assessed for its mass or shape. Mass orshape of the viral vector particle can be determined using techniquesinvolving point excitation and/or point collection of emissions,combinations of reflective mirrors, or CCD cameras, which are known inthe art. Such methods can provide an additional technique fordetermination of the quality and/or number of the viral vector particlesin the medium.

The invention also provides a method of evaluating the viral vectorparticle content of a medium. In this respect, a medium, which can beany medium described herein, is provided and contacted with anexcitation radiation having an excitation wavelength such that if aviral vector particle is in the medium an intrinsically fluorogenicportion of the viral vector particle will emit radiation having anemission wavelength at about 560-590 nm. The viral vector particlecontent (e.g., adenoviral vector content) of the medium is thenevaluated by determining whether the medium emits radiation at about560-590 nm.

This method can be used to detect the presence or absence of any viralvector particle (such as an adenoviral vector particle) which includesan intrinsically fluorogenic portion that produces fluorescent emissionshaving an emission wavelength at about 560-590 nm, more precisely about570-580 nm, and even more precisely about 574 nm, when contacted with anexcitation energy. Any suitable excitation energy described herein canbe used. Preferably, the excitation energy maximizes viral vectorparticle-associated fluorescent emissions at about 574 nm. The methodalso can be practiced with components of such viral vectors, such as aviral protein or substantial homolog thereof, which has an emissionwavelength of about 560-590 nm.

It has been discovered that viral vector particles can be detected bysuch emission wavelengths which are significantly higher than theemission wavelengths associated with fluorogenic amino acids (e.g.,tryptophan) or nucleic acid bases (e.g., uracil). Moreover, detection atsuch wavelengths offers greater selectivity and possibly greatersensitivity in detection.

As described in connection with other aspects of the present invention,the medium can be provided by contacting a chromatography resin with aviral vector particle-containing composition and eluting the viralvector particle from the chromatography resin. Preferably, if a viralvector particle is in the composition, it will elute at a known (i.e.,standard) time. The portion of the composition which would contain aviral vector particle, if present, is used as the medium, therebyverifying that any detected emission wavelengths at about 560-590 nmoriginate from the viral vector particle rather than some otherfluorogenic molecule.

When the medium contains a viral vector particle, the method can furtherinclude quantifying the number of viral vector particles, by using thequantification techniques described herein. Thus, for example, theintensity of the emitted radiation associated with the intrinsicallyfluorogenic portion of the viral vector particle can be determined, andthe number of viral vector particles in the medium can be quantified bycomparing the intensity of the detected radiation with a standardsignal.

The identification and/or quantification of viral vector particles(including the number of defective, empty, or otherwise damagedparticles) by fluorescence detection can be verified. For example, themethod can further include detecting the mass or shape of anyfluorescent molecule in the medium emitting radiation at about 560-590nm, using techniques described herein or otherwise known in the art.

The invention further provides a method of evaluating the adenoviralvector particle content of a medium through fluorescence detection. Ithas been discovered that wild-type adenoviral vectors include a capsidwhich consists essentially of intrinsically fluorogenic proteins. Thus,any adenoviral vector containing a wild-type capsid protein, or asubstantial homolog thereof, will include an intrinsically fluorogenicportion. In view of these fluorogenic properties, adenoviral vectorparticles are particularly well suited for fluorescence detection.

In this respect, a medium, which can be any medium as described herein,is contacted with an excitation radiation such that, if an adenoviralvector particle is in the medium, an intrinsically fluorogenic portionof the adenoviral vector particle will emit radiation having an emissionwavelength indicative (i.e., characteristic) of an adenoviral vectorparticle. The excitation radiation can have any suitable excitationwavelength. Typically, adenoviral vectors are associated with excitationwavelengths of about 220-240 nm, about 270-290 nm, or both; moreprecisely about 230-240 nm, about 280-290 nm, or both; and even moreprecisely about 235 nm, about 284 nm, or both.

The adenoviral vector particle content of the medium is evaluated bydetermining whether radiation having an emission wavelength indicativeof the presence or absence of an adenoviral vector particle is emittedupon contacting the medium with the excitation radiation. Thisdetermination is arrived at by comparing the detected emissionwavelengths with emission wavelengths normally associated withadenoviral vectors. Any suitable wavelength or combination ofwavelengths indicative of an adenoviral vector can be used. Typically,adenoviral vector-associated emission wavelengths include wavelengths atabout 320-340 nm, about 560-590 nm, or both; more precisely about328-332 nm, about 570-580 nm, or both; and even more precisely at about330 nm, about 574 nm, or both.

As described in connection with other aspects of the present invention,the medium can be provided by contacting a chromatography resin with anadenoviral vector-containing composition and eluting the adenoviralvector from chromatography resin. Preferably, if an adenoviral vectorparticle is in the composition, it will elute at a known (i.e.,standard) time. The portion of the composition which would contain anadenoviral vector particle, if present, is used as the medium.

When the medium contains an adenoviral vector particle, the method canfurther include quantifying the number of adenoviral vector particles,by using the quantification techniques described herein. Thus, forexample, the intensity of the emitted radiation associated with theintrinsically fluorogenic portion of the adenoviral vector particle canbe determined, and the number of adenoviral vector particles in themedium can be quantified by comparing the intensity of the detectedradiation with a standard signal. Preferably, quantification ofadenoviral vector particles is performed using an excitation wavelengthof about 270-295 nm (e.g., 284 nm) and an emission wavelength of about560-590 nm (e.g. 574 nm).

The identification and/or quantification of adenoviral vector particles(including the number of damaged particles) by fluorescence detectioncan be verified. For example, the method can further include detectingthe mass or shape of any fluorescent molecule in the medium emittingradiation at wavelengths indicative of adenoviral vector particles,using techniques described herein or otherwise known in the art.

The inventive methods also can include separation and identification ofthe intrinsically fluorogenic portion of the viral vector particle orcomponents thereof The fluorogenic portion and its components can beseparated by any suitable method. For example, the components of achemically disassociated viral vector particle can beelectrophoretically separated, based on size and/or charge. Preferably,separation of the fluorogenic portion or its components is performed byreverse phase chromatography. These separated components can besubjected to fluorescence detection to identify and/or characterize thefluorogenic portion of the viral vector particle.

The invention additionally provides a method of evaluating theintrinsically fluorogenic adenoviral structural protein content of amedium. An intrinsically fluorogenic adenoviral structural protein isany adenoviral protein that is a wild-type adenoviral protein or asubstantial homolog, which is intrinsically fluorogenic, and whichnormally forms a part of, or which can be made a part of, an adenoviralcapsid. Such proteins can be obtained, for example, by performingreverse phase chromatography, or other separation methods discussedherein or known in the art, on an adenoviral vector particle.Alternatively, such proteins can be produced by any other suitabletechnique (e.g., by recombinant DNA technology).

In this respect, a medium is provided, which can be any medium discussedherein, and contacted with an excitation radiation having an excitationwavelength, such that if an intrinsically fluorogenic adenoviralstructural protein is in the medium it will emit radiation having anemission wavelength characteristic of an intrinsically fluorogenicadenoviral structural protein. The adenoviral protein content of themedium is evaluated by determining whether radiation having an emissionwavelength characteristic of an intrinsically fluorogenic adenoviralstructural protein is emitted from the medium.

The medium can be provided by the use of chromatography to separate acomposition containing an intrinsically fluorogenic adenoviralstructural protein, such that an eluted portion of the composition willcontain an adenoviral vector structural protein, if present, asdescribed herein. Moreover, the method can include quantification of thenumber of adenoviral structural proteins in the medium. Theintrinsically fluorogenic adenoviral structural protein can be part of alarger complex, for example, a complex of proteins, or even part of adifferent type of vector.

Numerous alternative and equivalent techniques and devices to thosedescribed herein as useful in the context of the present invention arepossible. Several of such techniques and devices are known in the art,and are described in, for example, Brand, L. and Johnson, M. L., Eds.,Fluorescence Spectroscopy (Methods in Enzymology, Volume 278), AcademicPress (1997); Cantor and Schimmel, Biophysical Chemistry, W. H. Freeman& Co. (New York) (11th Printing 1998), Dewey, T. G., Ed., Biophysicaland Biochemical Aspects of Fluorescence Spectroscopy, Plenum Publishing(1991); Guilbault, G. G., Ed., Practical Fluorescence, Second Edition,Marcel Dekker (1990); Lakowicz, J. R., Ed., Topics in FluorescenceSpectroscopy: Techniques, Volumes 1-5 (1991); Plenum Publishing;Lakowicz, J. R., Principles of Fluorescence Spectroscopy, SecondEdition, Plenum Publishing (1999); and Sharma, A. and Schulman, S. G.,Introduction to Fluorescence Spectroscopy, John Wiley and Sons (1999).

EXAMPLES

The following examples further illustrate the present invention butshould not be construed as in any way limiting its scope.

Example 1

This example demonstrates the identification of excitation and emissionwavelengths for the intrinsically fluorogenic portion of a viral vectorparticle.

A 100 μl solution containing approximately 1.3×10⁹ anion exchangechromatography-purified, wild-type adenovirus particles (serotype 5) wasobtained. No fluorescent dyes or fluorophores were in, or added to, thesolution. The solution was placed in a Hewlett Packard 1100 scanningFluorescence Detector, equipped with a xenon flash excitation radiationsource and a PMT detector. The solution was scanned to determine whetherthe adenovirus particles were intrinsically fluorogenic by contactingthe solution with UV radiation emitted from the xenon flash lamp atwavelengths between 220 nm and 250 nm. An emission peak was detectedwhen the solution was contacted with excitation radiation having anexcitation wavelength at about 235 nm. Thus, it was determined that theadenovirus particles contain a naturally-occurring intrinsicallyfluorogenic portion.

Emission wavelengths then were scanned by fixing the excitationradiation at 235 nm (+/−10 nm), contacting the solution with theexcitation radiation, and detecting whether fluorescent emissions havingemission wavelengths of between 300 nm and 600 nm were produced. Aprominent emission wavelength was observed at about 330 nm.

Excitation wavelengths were re-scanned by fixing detected emissionwavelengths at 330 nm and scanning excitation wavelengths from 200 nm to300 nm. Two excitation wavelengths, one at about 234 nm and another atabout 284 nm, which resulted in significant fluorescent emissions at 330nm, were observed.

Emission wavelengths were then re-scanned by fixing the excitationwavelength at 281 nm (+/−10 nm) and detecting whether fluorescentemissions having emission wavelengths of between 300 nm and 700 nm wereproduced. Two emission wavelengths were observed: one at about 330 nm,and, surprisingly, a second emission wavelength at about 574 nm, whichis well above emission wavelengths associated with aromatic amino acids(282-348 nm), pyrimidine nucleotide bases (260-275 nm), and purinenucleotide bases (260-267 nm). Fluorescent emissions at the 574 nmemission wavelength were determined to be more selective for theadenovirus particles (i.e., emissions at this wavelength tended to beassociated with less background fluorescence and/or undesired excitationof other molecules in the solution) and resulted in significantly higheremission intensity than at 330 nm, indicating that this emissionwavelength was the optimum wavelength, and, thus, also capable ofproviding the most sensitive detection.

The results of these experiments demonstrate how excitation wavelengthsand emission wavelengths can be determined for a viral vector particlecontaining an intrinsically fluorogenic portion, such as an adenoviralvector particle. The results also demonstrate that viral vectorparticles which are associated with more than one excitation emissionwavelength can be detected using the method of the invention, and thatsuch wavelengths can be exploited to provide more sensitive and/or moreselective viral vector particle detection. Furthermore, these resultsdemonstrate that viral vector particles having an emission wavelength ofbetween 560-590 nm can be detected by fluorescent detection.

Example 2

This example demonstrates the relationship between emission intensityand total number of adenoviral vector particles in a medium.

A medium containing 3.84×10⁹ unmodified serotype 5 adenovirus particleswas subjected to six repeated 1:3 serial dilutions. At each dilution ofthe medium, three 100 μl samples of the diluted medium were obtained andsubjected to excitation radiation at 235 nm and fluorescence detectionusing a Hewlett Packard 1100 Fluorescence Detector, as described inExample 1. The excitation radiation wavelength was set at 235 nm.

Emission radiation having an emission wavelength of 330 nm and thecorresponding intensity of the emission wavelength for each of thesamples was detected by a PMT contained in the fluorescence detector.Intensity was measured in relative light units (RLUs). An emissionspectrum corresponding to detected emission wavelengths and intensitieswas produced. The area under each peak in the emission spectrum wasintegrated by ChemStation 3D version 8.0 (Hewlett Packard). Integratedpeak areas for each sample at each dilution were averaged except forresults which varied more than 5% from the mean peal area at a givendilution which were not considered. The results of these experiments arepresented in Table 2.

TABLE 2 Adenovirus Particle Number and Fluorescence Intensity EstimatedNumber of Adenovirus Particles in the Diluted Medium FluorescenceIntensity (RLU) 5.27 × 10⁶ 9.6 1.58 × 10⁷ 26 4.74 × 10⁷ 84 1.42 × 10⁸273 4.27 × 10⁸ 876 1.28 × 10⁹ 2525

These results were plotted on a graph of the number of adenovirusparticles in the diluted medium versus the intensity of the fluorescenceintensity. The plotted data formed a line. The linear nature of therelationship between viral vector particle number and fluorescentintensity was confirmed by linear regression analysis. The regressioncoefficient was determined to be 0.9998.

These results show that mediums containing viral vector particles whichinclude an intrinsically fluorogenic portion exhibit fluorescenceintensity in a linear relationship to the number of viral vectorparticles in the medium, and, thus, are subject to quantification byfluorescent detection. The results further demonstrate that this linearrelationship extends across a wide range of total viral vector particlenumbers (e.g., from about 5.27×10⁶ particles to about 1.28×10⁹particles). Thus, the present invention provides a method of quantifyingthe number of viral vector particles in a medium by fluorescentdetection.

Example 3

This example demonstrates numerous aspects of the present inventivemethod including the increased sensitivity of the inventive method overpresently used UV spectrophotometry-based techniques, as well as therelative quantification of intact adenovirus particles and the relativequantification of defective and empty adenovirus particles byfluorescence detection in various mediums.

Cells infected with unmodified serotype 5 adenovirus were harvested toobtain a crude cell lysate using standard techniques. A sample of thecrude cell lysate was obtained.

Two additional samples were prepared as follows: Two aliquots of theremaining cell lysate were obtained and subjected to purification bycontact with an anion exchange high performance liquid chromatographycolumn (AE-HPLC) as described in International Patent Application WO99/54441, or by separation on a cesium chloride density gradientrepeated three times (i.e., triple, or 3×, CsCl density gradientpurification). Volumes equal to the crude lysate sample were obtainedfor both purified samples.

Yet two more samples were prepared as follows: Another aliquot of thecell lysate was subjected to AE-HPLC purification followed by one time(i.e., 1×) CsCl density gradient purification. After 1×CsCl densitygradient purification, two distinct bands on the AE-HPLC column eluant(an upper and lower band), corresponding to the purified AE-HPLC eluantwere observable. Samples extracted from each band, in a volume equal tothe aforementioned samples, were obtained to provide an upper bandsample and a lower band sample. Mass spectrometry analysis determinedthat the upper band sample contained higher quantities of incompletelyprocessed (i.e., empty and defective) adenovirus particles than thelower band sample, and even more defective and empty particles than thecrude cell lysate.

Each of the aforementioned five samples was divided into three equalvolumes. The equal volumes were separately injected into an analyticalchromatography column which was connected to both a UV spectrophotometerand the fluorescence detector described in Example 1. Thus, the portionsof the samples eluted from the analytical column were subjected to UVspectrophotometry-based absorbance analysis as well as excitation andfluorescence detection almost immediately following elution. The time ofelution from the analytical column and absorbance of fluorescencedetection were determined. Elution times for viruses detected byabsorbance and excitation varied by 1 second or less.

Absorbance quantification was performed at 260 nm for each portioneluted from the analytical column using standard techniques. The eluantobtained from the first equal volume of each sample was contacted withexcitation radiation having an excitation wavelength (Ex) of 235 nm.This was followed by fluorescence detection at the 330 nm emissionwavelength (Em). The eluants obtained from the second and third equalvolumes of each sample were contacted with excitation radiation at 284nm, which was followed by fluorescence detection at either the 330 nm or574 nm emission wavelength, respectively. Fluorescent intensity in allcases was measured in relative light units.

Absorbance and fluorescence intensities for adenovirus particles in eacheluted portion were separately plotted against time of elution toprovide two dimensional graphs containing absorbance or emission peaks.Integrated peak areas for absorbance, and for fluorescence intensitycorresponding to each of the excitation and emission wavelengthcombinations used (i.e., 235 nm Ex: 330 nm Em, 284 nm Ex: 330 nm Em, and284 nm Ex: 574 nm Em), were determined. Normalized values for eachportion were determined by dividing the integrated area of thefluorescence peaks by the integrated area of the absorbance peaks. Theresults of these calculations are shown in Table 3.

TABLE 3 Normalized Fluorescence/Absorbance Values for Adenoviral VectorParticles in Various Mediums Fluorescence Fluorescence FluorescenceIntensity Peak Intensity Peak Intensity Peak Area (Ex = 235 Area (Ex =284 Area (Ex = 284 nm:Em = 330 nm:Em = 330 nm:Em = 574 nm)/Absorbancenm)/Absorbance nm)/Absorbance Peak Area (260 Peak Area (260 Peak Area(260 nm) nm) nm) Crude cell lysate 16.0 22.3 14.4 AE-HPLC purified 16.022.3 20.5 lysate 3x CsCl density- 14.3 20.2 19.6 gradient purifiedlysate AE-HPLC purified + 14.2 19.5 19.1 1x CsCl density gradientpurification (lower band) AE-HPLC purified + 15.0 21.1 13.1 1x CsCldensity gradient purification (upper band)

The results of these experiments are significant in many respects.First, the normalized values in Table 3 are indicative of the relativesensitivity of the inventive method as compared to UVspectrophotometry-based absorbance analysis. Particularly, as can beseen from the data set forth in Table 3, the fluorescence detectionmethods of the present invention are more sensitive than detectionmethods based on UV absorbance.

Second, the results of these experiments demonstrate quantification ofthe number of adenovirus particles in a medium by the inventive method.For example, the lower band sample, which contained only a portion ofthe eluant obtained from the AE-HPLC purification step, exhibitedsignificantly lower normalized values than crude cell lysate and AE-HPLCpurified samples at the 330 nm emission wavelength. These lowernormalized values reflect lower fluorescence intensity at the 330 nmemission wavelength emitting from the lower band sample, and,consequently, reflect a smaller number of viral particles in the lowerband sample versus the crude cell lysate and AE-HPLC purified samples,as predicted. Thus, these results confirm that the present inventionprovides a method for relatively quantifying the number of viral vectorparticles in a medium by fluorescence detection. The consistentnormalized values obtained at 330 nm emission wavelengths for theAE-HPLC purified sample and crude cell lysate sample indicate thatnearly all of the particles were retained by the AE-HPLC purificationtechnique and corroborate the ability of the inventive method toquantify the number of viral particles in the medium.

Third, these results demonstrate the relative quantification ofdefective and empty viral vector particles in a medium by fluorescencedetection. For example, as seen in Table 3, the crude cell lysate sampleexhibited a significantly lower normalized value, and, thus, lowerfluorescence intensity, at the 574 nm emission wavelength, than theAE-HPLC purified sample. The upper band sample exhibited an even moresignificant decrease in normalized value at the 574 nm emissionwavelength as compared to the AE-HPLC purified sample. The decrease influorescence intensity at the 574 nm emission wavelength observed in thecrude cell lysate and upper band samples versus the AE-HPLC purifiedsample corresponds to the higher number of defective and empty particlesin these samples, as confirmed by mass spectrometry experiments.Similarly, the relatively higher fluorescence intensity at the 547 nmemission wavelength observed for the lower band sample versus theAE-HPLC purified sample corresponds to the lower number of defective andempty particles in this sample, as also confirmed by mass spectrometryexperiments.

Using fluorescent emissions at the 574 nm emission wavelength from theupper band as a standard signal, and by performing a linear regressionanalysis, the relative percentage of empty and defective particles inother samples was determined. For example, by performing such ananalysis it was determined that the AE-HPLC sample contained about 11.5%of the number of empty and defective particles contained in the upperband sample.

The results of these experiments also confirm that the 330 nm emissionwavelength is relative damage-insensitive for adenovirus particles andthat the 574 nm emission wavelength is a damage-sensitive emissionwavelength for adenovirus particles, which is associated with anintensity shift. The relative consistency of fluorescence intensity atthe 330 nm emission wavelength for the various samples confirms thatthis wavelength is a relatively damage-insensitive wavelength. Therelative variance of fluorescence intensity at the 574 nm emissionwavelength between the various samples known to differ as to thequantity of damaged viral vector particles confirms that this wavelengthis a damage-sensitive wavelength for adenovirus particles. Thus, thenormalized value (or fluorescence intensity) observed at the 330 nmemission wavelength can be used to calculate the total number ofparticles, and the value obtained at the 574 nm emission wavelength candetermine what proportion of those particles are damaged.

This experiment demonstrates that the present inventive method can beused to quantify the number of viral vector particles in a medium and/orquantify the number of damaged (e.g., defective and empty) viral vectorparticles in a medium.

Example 4

This example demonstrates the separation and identification of theintrinsically fluorogenic components of adenovirus particles and theevaluation of the intrinsically fluorogenic adenoviral structuralprotein content of a medium.

A first solution containing 1.9×10⁹ intact wild-type serotype 5adenovirus particles and a second solution containing 3.5×10¹⁰ intactwild-type serotype 5 adenovirus particles were obtained using standardtechniques. Each solution was subjected to C4 reverse phasechromatography without disassociation of the adenovirus particles priorto contacting the reverse phase column with the solutions. Theadenovirus particles were separated into their constituent molecules bycontact with the reverse phase chromatography resin, and the separatedcompounds were eluted from the resin. The time of elution of thecomponents was determined.

After elution, the eluted portion of the second solution was subjectedto UV spectrophotometry at 214 nm to identify the separated componentsof the adenovirus particles. The resulting absorbance was plotted on agraph against the time of elution of the components. Peak area wasdetermined to identify the proportion of the total adenovirus proteincontent corresponding to each detected component.

Similarly, after elution, the eluted portion of the first solution wassubjected to excitation radiation having an excitation wavelength at 235nm and fluorescent detection for emissions having an emission wavelengthat 330 nm using the techniques described in Example 1 to determine whichof the identified components were intrinsically fluorescent.Fluorescence intensity was plotted on a graph against the time ofelution of the fluorescent components. Peak area was determined toidentify the proportion of the fluorescence intensity of eachfluorogenic component to the combined fluorescence for all components,thereby enabling determination of the relative fluorescence of theintrinsically fluorogenic components.

Significant absorbance peaks were observed for components eluted fromthe resin at about 9.5 minutes, 10 minutes, 10.5 minutes, 11.5 minutes,12 minutes, 12.5 minutes, 14 minutes, 15 minutes, 22 minutes, and 24minutes. These peaks were correlated with fluorescence peaks observedfor components eluted from the resin at about 2.5 minutes, 12 minutes,13.5 minutes, 15.5 minutes, 16 minutes, 18 minutes, 19 minutes, 26minutes, and 28 minutes. Other peaks were determined to be caused bydefective or empty adenovirus particles or buffers in the solution andwere not further analyzed. Correlation of the detected components wasconfirmed, and molecules identified, by enzymatic digestion and massspectrometry analysis (including amino acid sequencing) of the elutedcomponents for both solutions.

By comparing the resulting absorbance and fluorescence spectrums, it wasdetermined that the absorbance peak corresponding to the componenteluted at 9.5 minutes was not represented by a corresponding peak in thefluorescence spectrum. By mass spectrometry analysis it was determinedthat this component consists of the N-terminal portion of the adenovirusmajor core protein (protein VII). In contrast, each of the otherdetected components were determined to fluoresce when contacted withexcitation radiation at 235 nm.

Comparisons were made between the areas of the absorbance peaks and thefluorescence peaks to determine the relative component content of theelution and its proportional contribution to the total fluorescence ofthe solution. By making such comparisons the fluorescent qualities ofthe components was relatively determined. For example, the adenovirusprotein hexon, which eluted at 22 minutes in solution, was determined byits absorbance peak area to make up 52% of the total protein content ofthe adenovirus particles (consistent with published figures), whereasits fluorescent emission contribution was about 75% of the totaldetected fluorescence of the components. Thus, hexon was identified as astrong intrinsically fluorogenic adenoviral protein. Other intrinsicallyfluorogenic adenovirus structural proteins identified by theseexperiments include the adenovirus fiber and penton proteins. Similaremission patterns were seen in other experiments performed usingexcitation radiation having an excitation wavelength of 284 nm andfluorescence detection at 330 nm.

These results demonstrate that components of intrinsically fluorogenicportions of viral vector particles can be separated and evaluated byfluorescence detection. Moreover, these results demonstrate that theadenoviral vector structural protein content of a medium can bedetermined by fluorescence detection.

All references, including publications, patent applications and patents,cited herein are hereby incorporated by reference to the same extent asif each reference were individually and specifically indicated to beincorporated by reference and were set forth in its entirety herein. Theuse of the terms “a” and “an” and “the” and similar referents in thecontext of describing the present invention (especially in the contextof the following claims) are to be construed to cover both the singularand the plural, unless otherwise indicated herein or clearlycontradicted by context. The use of the terms “including,” “having,”“comprising,” “containing, ” and similar terms are to be construed asopen-ended terms (i.e., meaning “including, but not limited to”) unlessotherwise indicated. Recitation of ranges of values herein are merelyintended to serve as a shorthand method of referring individually toeach separate value falling within the range, unless otherwise indicatedherein, and each separate value is incorporated into the specificationas if it were individually recited herein. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illustrate the present invention and is notintended as a limitation on the scope of the claimed invention. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

The foregoing is an integrated description of the invention as a whole,not merely of any particular element or facet thereof. The descriptiondescribes “preferred embodiments” of this invention, including the bestmode known to the inventors for carrying it out. Upon reading theforegoing description, variations of those preferred embodiments maybecome apparent to those of ordinary skill in the art. The inventorsexpect skilled artisans to employ such variations as appropriate, andthe inventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is possible unless otherwise indicated herein or otherwiseclearly contradicted by context.

What is claimed is:
 1. A method of evaluating the content of aparticular intrinsically fluorogenic adenoviral structural protein in acomposition, the method comprising: (a) contacting a composition with achromatography resin, (b) eluting at least a portion of the compositionfrom the chromatography resin such that if the particular intrinsicallyfluorogenic adenoviral structural protein is in the composition it willelute at a known time and in substantial absence of other intrinsicallyfluorogenic adenoviral structural proteins, (c) obtaining the portion ofthe composition which will contain the particular intrinsicallyfluorogenic adenoviral structural protein, if present in thecomposition, to provide a medium, (d) contacting the medium with anexcitation radiation comprising an excitation wavelength of about 284,such that if the particular intrinsically fluorogenic adenoviralstructural protein is in the medium it will emit radiation comprising anemission wavelength of about 330 nm, and (e) evaluating the content ofthe particular intrinsically fluorogenic adenoviral structural proteinin the composition by determining whether radiation comprising anemission wavelength of about 330 nm is emitted from the medium.
 2. Themethod of claim 1, wherein the particular intrinsically fluorogenicadenoviral structural protein is hexon.
 3. The method of claim 1,wherein the particular intrinsically fluorogenic adenoviral structuralprotein is fiber.
 4. The method of claim 1, wherein the particularintrinsically fluorogenic adenoviral structural protein is penton base.5. A method of evaluating the content of a particular intrinsicallyfluorogenic adenoviral structural protein in a composition, the methodcomprising: (a) contacting a composition with a chromatography resin,(b) eluting at least a portion of the composition from thechromatography resin such that if the particular intrinsicallyfluorogenic adenoviral structural protein is in the composition it willelute at a known time and in substantial absence of other intrinsicallyfluorogenic adenoviral structural proteins, (c) obtaining the portion ofthe composition which will contain the particular intrinsicallyfluorogenic adenoviral structural protein, if present in thecomposition, to provide a medium, (d) contacting the medium with anexcitation radiation comprising an excitation wavelength of about 235,such that if the particular intrinsically fluorogenic adenoviralstructural protein is in the medium it will emit radiation comprising anemission wavelength of about 330 nm, and (e) evaluating the content ofthe particular intrinsically fluorogenic adenoviral structural proteinin the composition by determining whether radiation comprising anemission wavelength of about 330 nm is emitted from the medium.
 6. Themethod of claim 5, wherein the particular intrinsically fluorogenicadenoviral structural protein is hexon.
 7. The method of claim 5,wherein the particular intrinsically fluorogenic adenoviral structuralprotein is fiber.
 8. The method of claim 5, wherein the particularintrinsically fluorogenic adenoviral structural protein is penton base.